JP2008539710A - Thermophilic microorganism with inactivated lactate dehydrogenase (LDH) gene for ethanol production - Google Patents

Abstract

A mutant thermophilic microorganism is produced that has been modified to inactivate the lactate dehydrogenase gene of the wild-type microorganism. The mutant microorganism is used in ethanol production and utilizes a C ₃ , C ₅ or C ₆ sugar as a substrate.

Description

  The present invention relates to ethanol production as a bacterial fermentation product. In particular, the present invention relates to ethanol production by thermophilic bacteria.

  Bacterial metabolism can occur through a variety of different mechanisms, depending on the bacterial species and environmental conditions. Heterotrophic bacteria, including all pathogens, are compounds that gain energy from the oxidation of organic compounds and most commonly oxidize carbohydrates (especially glucose), lipids, and proteins. Biooxidation of these organic compounds by bacteria results in the synthesis of ATP as a source of chemical energy. The process also allows for the production of simpler organic compounds that are required by bacterial cells for biosynthetic reactions. A common method by which the bacteria metabolize the appropriate substrate is glycolysis, which is a series of reactions that convert glucose to pyruvate resulting in the production of ATP. In producing metabolic energy, the fate of pyruvate varies depending on bacteria and ambient conditions. There are three main reactions of pyruvic acid.

  First, under aerobic conditions, many microorganisms use the citrate cycle to generate energy, and the conversion of pyruvate to acetyl coenzyme A is catalyzed by pyruvate dehydrogenase (PDH).

  Second, under anaerobic conditions, ethanol producing organisms can be obtained by decarboxylation of pyruvate to acetaldehyde catalyzed by pyruvate decarboxylase, followed by reduction of acetaldehyde to ethanol by NADH catalyzed by alcohol dehydrogenase. Alcohol fermentation can be performed.

  Third, the conversion of pyruvate to lactic acid, which occurs through a catalytic reaction with lactate dehydrogenase (LDH).

  Considerable to use microorganisms to produce ethanol using either microorganisms that naturally undergo anaerobic fermentation or that perform anaerobic fermentation through the use of recombinant microorganisms incorporating the pyruvate decarboxylase and alcohol dehydrogenase genes Is interested in. Although some success has been achieved in producing ethanol using these microorganisms, fermentation is often hampered by high concentrations of ethanol, especially if the microorganisms have only a low level of ethanol resistance.

  Thermophilic bacteria are recommended for ethanol production, and the use of thermophilic bacteria has the advantage that fermentation can occur at high temperatures. High temperature fermentation allows the ethanol produced to be removed as steam at temperatures above 50 ° C. This makes it possible to carry out fermentation using high sugar concentrations. However, finding a suitable thermophilic bacterium that can efficiently produce ethanol is a problem.

  WO01 / 49865 discloses a gram positive bacterium for ethanol production transformed with a heterologous gene encoding pyruvate decarboxylase and having wild type alcohol dehydrogenase function. The bacterium is a thermophilic Bacillus and the bacterium can be modified by inactivating the lactate dehydrogenase gene using transposon insertion. The bacteria disclosed in WO01 / 49865 are all derived from a Bacillus LLD-R strain, ie a sporulation-deficient strain that occurs spontaneously from culture, where the ldh gene is either spontaneously generated or chemically abrupt. Inactivated by mutagenesis. LN and TN strains are disclosed as modified derivatives of the LLD-R strain. However, all strains contain a Hae type III restriction system, which prevents plasmid transformation and consequently, transformation into unmethylated DNA.

  WO01 / 85966 discloses microorganisms prepared by in vivo methylation to overcome the limitation problem. This requires transforming HaeIII methyltransferase from Haemophilus aegyptius into LLD-R, LN, and TN strains. However, the LLD-R, LN, and TN strains are unstable mutations and spontaneously return to lactic acid producing wild type strains, especially at low pH and high sugar concentrations. This changes the fermentation product from ethanol to lactic acid, making the strain unsuitable for ethanol production.

  WO02 / 29030 discloses that LLD-R and its derivatives contain a natural insertion element (IE) in the coding region of the ldh gene. Transfer of IE to the ldh gene (and from that gene) and subsequent gene inactivation is unstable and reversion occurs. The proposed solution for this was to incorporate plasmid DNA into the IE sequence.

  As a result, in the art, the production of microorganisms for ethanol production involves modifying laboratory-produced chemically mutated Bacillus microorganisms and treating these microorganisms with in vivo methylation methods. And further relies on modifying the microorganism to incorporate plasmid DNA into the IE sequence. The method is complex, uncertain and there are regulatory issues as to how the strain can be used.

  As a result, there is a need for improved microorganisms for ethanol production.

  In the first aspect of the invention, the thermophilic microorganism is modified to allow high ethanol production, the modification being an inactivation of the lactate dehydrogenase gene of a wild type thermophilic microorganism.

  In a second aspect of the invention, the microorganism is the microorganism deposited under NCIMB Deposit No. 41275.

In a third aspect of the invention, the method for ethanol production comprises culturing a microorganism according to the definition given above under suitable conditions in the presence of a C ₃ , C ₅ , or C ₆ sugar. .

In a fourth aspect of the invention, the plasmid is a plasmid as defined herein as pUB190-ldh (deposited as NCIMB Deposit No. 41276).
In a fifth aspect of the invention, the thermophilic microorganism comprises a plasmid defined herein as pUB190 (FIG. 4).

The present invention is based on modifying wild type thermophilic microorganisms to prevent the expression of lactate dehydrogenase gene.
Inactivating the lactate dehydrogenase gene helps prevent the degradation of pyruvate into lactate, and as a result (under appropriate conditions) pyruvate is ethanolized using pyruvate decarboxylase and alcohol dehydrogenase. To break down into Preferably, the lactate dehydrogenase gene is hindered by a deletion in the gene or a deletion of the gene.

  The wild-type microorganism may be any thermophilic microorganism, but is preferably a microorganism of Bacillus spp. In particular, it is preferable that the microorganism is a Geobacillus species, in particular, Geobacillus thermoglucosidasius.

  The microorganism selected for modification is referred to as “wild type”, that is, the microorganism is not a mutant produced in the laboratory. Microorganisms may be isolated from environmental samples that are predicted to contain thermophiles. Isolated wild-type microorganisms have the ability to produce ethanol, but if not modified, lactic acid appears to be the major fermentation product. Isolates are selected for their ability to grow on hexose and / or pentose sugars and their oligomers at elevated temperatures.

It is preferred that the microorganism has certain desirable properties that allow it to be used in the fermentation process. The microorganism should preferably not have a restriction system, thereby avoiding the need for in vivo methylation. In addition, the microorganism should be stable with at least 3% ethanol and should have the ability to utilize C ₃ , C ₅ , and C ₆ sugars (or oligomers thereof) including cellobiose and starch as substrates. It is. It is preferable that the microorganism can be transformed with high frequency. In addition, the microorganism should have a growth rate in continuous culture (typically 0.3 OD ₆₀₀ ) that supports a dilution rate of 0.3 h ⁻¹ and higher.

  The microorganism is thermophilic and grows in the temperature range of 40 ° C to 85 ° C. Preferably, the microorganism grows in a temperature range of 50 ° C to 70 ° C. Further, the microorganism grows under conditions of pH 7.2 or lower, particularly conditions of pH 6.9 to pH 4.5.

  The microorganism may be a spore former or may not be able to sporulate. The success of the fermentation process does not necessarily depend on the ability of the microorganism to sporulate, but in certain circumstances, if it is desired to use the microorganism as an animal feed at the end of the fermentation process, use spore-forming bacteria. Is preferred. This is due to the ability of spore-forming bacteria to provide good immune stimulation when used as animal feed. Sporulating microorganisms also have the ability to initiate sporulation during fermentation so that they can be isolated without the need for centrifugation. Thus, microorganisms can be used in animal feed without the need for complicated or expensive separation methods.

  The nucleic acid sequence of lactate dehydrogenase is currently known. Using the sequence, one skilled in the art can target the lactate dehydrogenase gene to achieve inactivation of the gene through various mechanisms. It is preferred that the lactate dehydrogenase gene is inactivated by insertion of a transposon, or preferably by deletion of the gene sequence or part of the gene sequence. Deletions are preferred. This is because deletion can avoid problems with gene sequence reactivation, which often occurs when transposon inactivation is used. In a preferred embodiment, the lactate dehydrogenase gene is inactivated by integration of a temperature sensitive plasmid (plasmid pUB190-ldh), which achieves natural homologous recombination or integration between the plasmid and the microbial chromosome. Chromosomal integrants can be selected based on the resistance of the integrant to an antimicrobial agent (eg, kanamycin). Integration into the lactate dehydrogenase gene may occur by single crossover recombinant or double (or more) cross recombination.

  In a preferred embodiment, the microorganism comprises a heterologous alcohol dehydrogenase gene and a heterologous pyruvate decarboxylase gene. The expression of these heterologous genes results in the production of enzymes that direct metabolism so that ethanol is the main fermentation product. These genes may be obtained from microorganisms that typically lead to anaerobic fermentation, such as zymomonnas species, such as zymomonas mobilis.

  Methods for incorporating these genes into microorganisms are known, for example, Ingram et al., Biotech & BioEng, 1998; 58 (2 + 3): 204-214 and US5916787, the contents of which are Incorporated herein by reference. The gene may be introduced into a plasmid or may be integrated into the chromosome, as will be appreciated by those skilled in the art.

  The microorganisms of the present invention may be cultured under conventional medium conditions according to the selected thermophilic microorganism. The choice of substrate, temperature, pH, and other growth conditions can be selected based on known media requirements. See, for example, WO01 / 49865 and WO01 / 85966. The contents of each are incorporated herein.

  The invention will now be described by way of example in the following examples with reference to the accompanying drawings.

Inactivation of LDH gene
Example 1- Development of Single Cross Mutagenesis LDH Knockout Vector A portion of the approximately 800 bp ldh gene fragment was subcloned into the temperature sensitive delivery vector pUB190 (SEQ ID NO: 1) using HindIII and XbaI to yield a 7.7 kb The pUB190-ldh plasmid (Figure 4 and SEQ ID NO: 2) was obtained. The pUB190 plasmid has been deposited with NCIMB as described below. Ten putative E. coli JM109 (pUB190-ldh) transformants were confirmed by restriction enzyme analysis, and two cultures were used to obtain plasmid DNA for transformation. Cleavage of pUB190ldh with HindIII and XbaI releases the expected ldh fragment.

Using a kanamycin screen in which Geobacillus thermoglucosidasius 11955 was transformed with pUB190-ldh, transformants having all three test plasmids were obtained after 24-48 hours at 54 ° C. ldh transformants were isolated from Geobacillus thermoglucosidosis (Gt) 11955 and verified by restriction enzyme analysis using HindIII and XbaI.

Gene knockout was performed by integrating the LDH gene knockout temperature sensitive plasmid into the ldh gene on the chromosome.
Plasmid pUB190-ldh replicated at 54 ° C. on Gt11955 but not at 65 ° C. Sorting was maintained with kanamycin (kan) (12 μg / ml). The growth temperature was then increased to 65 ° C. (the plasmid was no longer replicated). Natural recombination or integration occurs between the plasmid and the chromosome. Chromosomal integrants were screened for resistance of the integrant to kanamycin. Since the homologous sequence is present on the plasmid, integration is made to the ldh gene. Targeted integration into the ldh gene occurred by a method known as single crossover recombination. The plasmid is incorporated into the ldh gene, resulting in an inactive ldh gene. Tandem repeats can occur when several copies of the plasmid are integrated.

Methodology and results Two different methods have been attempted to obtain integrants:
Method 1:
4 × 50 ml TGP (kan) cultures were grown at 54 ° C. for 12-18 hours. Cells are pelleted by centrifugation and suspended in 1 ml TGP. The suspension was plated on TGP (kan) plates (5 × 200 ml) and incubated overnight at 68 ° C. The integrants were removed and plated on a 50 square grid on a new TGP (kan) plate and incubated overnight at 68 ° C.

  Method 2: 1 × 50 ml TGP (km) culture was grown at 54 ° C. for 12-18 hours. 1 ml culture was used to inoculate 50 ml new TGP (kan) culture and the culture was grown at 68 ° C. for 12-18 hours. The next day the culture was pre-cultured on 50 ml fresh TGP (kan) culture and grown at 68 ° C. for an additional 12-18 hours. Cultures were plated on TGP (Km) plates and incubated overnight at 68 ° C. Grow to confluence on the plate. Single colonies were plated on a 50 square grid of fresh TGP (kan) plates and incubated overnight at 68 ° C.

Screening putative integrants were screened for ldh gene knockout using the following assay.
1) Plate sorting Replicate hundreds of integrants onto SAM2 plates (with kan) at 68 ° C. Lactic acid negative cells will produce less acid and will have a growth gain over wild type in fermentation media without buffer.
2) PCR selection Colony PCR is used to determine if the plasmid was integrated into the ldh gene. By selecting a primer adjacent to the integration site, it can be determined whether ldh gene integration has occurred (the PCR fragment for the insert is not amplified).
3) Lactate assay This assay determines whether integrants produce lactic acid when grown overnight in SAM2 (kan) at 68 ° C. The culture supernatant was tested for lactic acid concentration using Sigma lactic acid reagent for lactic acid determination. Lactic acid negative integrants were further characterized by PCR and evaluated for stability in fermentation vessels.

Electroporation protocol for Geobacillus thermoglucosidosis NCIMB 11955 Growing overnight cultures in TGP medium, adding an equal volume of 20% glycerol and aliquoting into 1 ml aliquots and in cryotubes at −80 ° C. A frozen stock of NCIMB 11955 was made by storing at. To inoculate 50 ml of TGP in a 250 ml conical flask, 1 ml of the stock was used and incubated at 55 ° C. and 250 rpm until the culture reached OD ₆₀₀ 1.4.

  The flask was incubated on ice for 10 minutes, then the culture was centrifuged for 20 minutes at 4 ° C. and 4000 rpm. The pellet was suspended in 50 ml ice-cold electroporation medium and centrifuged for 20 minutes at 4000 rpm. Three more washes are performed (1 × 25 ml and 2 × 10 ml), then the pellet is suspended in 1.5 ml ice-cold electroporation medium and divided into 60 μl aliquots.

  For electroporation, 1-2 μl of DNA was added to 60 μl of electrocompetent cells in an Eppendorf tube maintained on ice and mixed gently. This suspension was transferred to a pre-cooled electroporation cuvette (1 mm gap) and electroporated with 2500 V, 10 μF capacity and 600 Ω resistance.

  Immediately after pulsing, 1 ml of TGP was added, mixed, and the suspension was transferred to a screw-top tube and incubated in a shaking water bath at 52 ° C. for 1 hour. After incubation, the suspension is either sown directly (eg 2 × 0.5 ml) or centrifuged at 4000 rpm for 20 minutes, suspended in 200 μl to 500 μl of TGP and plated on TGP agar containing the appropriate antibiotic. It was.

Inactivation of LDH gene
Primers were designed based on the 11955LDH sequence available for double crossover mutation . The knockout strategy is based on creating internal deletions within the LDH gene by two approaches.

  Strategy 1: Two unique restriction sites near the middle of the LDH coding sequence were used to create the deletion. One large PCR product was generated from genomic DNA covering most of the available LDH sequences and cloned into the SmaI site of the multiple cloning site of pUC19. The pUC19 clone was then sequentially cut with BstEII and BsrGI and religated after Klenow cut to create an internal deletion between BstEII and BsrGI in the LDH gene.

  Strategy 2 (see Example 2): Cloning the LDH gene as two PCR products and introducing a NotI site into the oligo primer to allow the two PCR products to be ligated together to pUC19. A deletion was created in the middle of the LDH sequence. Two LDH genes with internal deletions were subcloned into three potential delivery systems for Geobacillus.

デリバリーベクターは、電気穿孔により１１９５５に形質転換された。

The delivery vector was transformed into 11955 by electroporation.

Genetic strategy information: Development of a delivery system for homologous recombination To create knockouts , deliver the mutated gene to the target organism and homologous recombination with the target "wild type" gene into the genome There is a need for an efficient system that is selected to incorporate. In principle, the efficient system can be achieved by introducing the DNA onto an E. coli vector without a Gram positive replicon but with a Gram positive selectable marker. This requires high transformation efficiency. The electroporation method developed for Geobacillus 11955 creates 3 × 10 ⁴ transformants per μg of DNA using pNW33N. Gram positive replicons are obtained from pBC1 in the BGSC catalog and from pTHT15 in the sequence database.

  The cat gene on pNW33N is used for selection in both E. coli and Geobacillus. A temperature sensitive mutant of pNW33N was generated by passaging the plasmid through the Stratagene XL1r ed mutagenized strain.

Example 2
Generation of LDH mutants by gene replacement An additional strategy was designed to generate stable mutations of the LDH gene in Geobacillus thermoglucosidasis NCIMB 1955 by gene replacement. The strategy involved making a 42 bp deletion near the middle of the coding sequence and inserting a 7 bp at this position that introduces a new Not1 restriction enzyme cleavage site. This inserted sequence was intended to cause a downstream frameshift mutation. The strategy involved creating deletions using two PCR fragments and using oligo primers that introduce a new NotI site. Primers were designed to be based on a partial sequence of the LDH coding region from 11955. The primer sequences used are shown below.

Genomic DNA Preparation Genomic DNA was prepared from 11955 and used as a template for PCR. Cells from 11955 20 ml overnight cultures grown in TGP medium at 52 ° C. were harvested by centrifugation at 4000 rpm for 20 minutes. The bacterial pellet was suspended in 5 ml STE buffer 0.3 M sucrose, 25 mM Tris-HCl and 25 mM EDTA and adjusted to pH 8.0 containing 2.6 mg lysozyme and 50 μl 1 mg / ml ribonuclease A. This was incubated for 1 hour at 30 ° C., then 5 mg proteinase K was added, and 50 μl of 10% SDS was added and incubated for 1 hour at 37 ° C. The lysed culture was then continuously extracted with an equal volume of phenol / chloroform followed by extraction with chloroform and then precipitation with isopropanol. After washing twice with ice-cold 70% ethanol, the DNA pellet was redissolved in 0.5 ml TE buffer.

Preparation of LDH deletion construct PCR was performed using a Robocycler Gradient 96 (Stratagene). The reaction conditions are as follows: cycle 1; denaturation at 95 ° C. for 5 minutes, annealing at 47 ° C. for 1 minute, extension at 72 ° C. for 2 minutes, cycle 2-30 times; denaturation at 95 ° C. for 1 minute, at 47 ° C. Anneal for 1 minute, extend at 72 ° C for 2 minutes, and further incubate at 72 ° C for 5 minutes. The enzyme used was an equal mixture of Pfu polymerase (Promega) and Taq polymerase (New England Biolabs, NEB). The buffer and dNTP composition and concentration used were those recommended for Pfu by the vendor. The PCR product obtained using genomic DNA from NCIMB 11955 as a template is purified by agarose gel electrophoresis and eluted from the agarose gel by using QIAquick Gel Extraction Kit (Qiagen). The purified PCR product was ligated into pUC19 (New England Biolabs) previously cut with SmaI and the ligation mixture was used to transform E. coli DH10B (Invitrogen). Ampicillin resistant colonies were selected and the contained plasmid was isolated and characterized by restriction enzyme analysis and the orientation of the insert was confirmed.

  Cleave the plasmid (pTM002) with fragment 2 inserted into pUC19 (with a new PstI site introduced on primer 4 closest to the PstI site in the multiple cloning site (mcs) of pUC19) with NotI and PstI did. The resulting fragment (approximately 0.4 kb) was ligated into the pUC19 plasmid (pTM001) with fragment 1 cut with NotI and PstI to linearize the plasmid. This ligation mixture was used to transform E. coli DH10B. Ampicillin resistant colonies were selected and the contained plasmid was isolated and characterized by restriction enzyme analysis. Identify the plasmid (pTM003) with the expected restriction enzyme pattern for the desired construct (LDH coding region has deletion and introduced NotI site) and sequence using M13mp18 reverse and forward primers Confirmed.

  The mutated LDH gene was excised from pTM003 by digestion with HindIII and EcoRI and purified by agarose gel electrophoresis, followed by elution from agarose (as an approximately 0.8 kb fragment) using the QIAquick gel excision kit. . The fragment was treated with Klenow polymerase (NEB, according to product instructions) to create blunt ends and introduced into the pUB190 vector. This was achieved by blunt end ligation with pUB190 linearized by cutting with XbaI, then Klenow, followed by gel purification as described above. The ligation mixture was used to transform E. coli SCS110 (Stratagene). Ampicillin resistant colonies were selected and the contained plasmid was isolated and characterized by restriction enzyme analysis. A plasmid (pTM014) having the expected restriction enzyme pattern for the cleavage construct was identified and used to transform NCIMB 11955 by electroporation using an electroporation protocol as described in Example 1. .

Generation and characterization of gene replacement LDH mutants by double crossover A double recombinant was obtained using the putative primary integrant of pTM014 obtained in this manner (gene replacement). This was achieved by serial passage of TM15 in TGP medium without kanamycin. Using 5 passage cultures, change from 8 hours at 54 ° C. to 16 hours at 52 ° C., and use 50 ml tubes (Falcon) with 5 ml TGP at 250 rpm and 1% transfer at each stage. Using. After these 5 passages, the resulting cultures were serially diluted in TGP and 100 μl samples were plated on TGP agar plates for incubation at 54 ° C. Colonies obtained on TGP agar containing 12 μg / ml kanamycin were identified using replica-plates to identify kanamycin sensitive colonies. After streaking a single colony on agar for purification, these kanamycin sensitive derivative strains were tested for lactic acid production and proved to be a mixture of LDH ⁺ and LDH ⁻ as expected. It was done. 1 of LDH ^- a derivative strain TM89 was characterized by further PCR and Southern blot.

Genomic DNA is produced from TM15 (first integrant) and TM89 (putative double recombinant LDH ⁻ ) and using primers 1 and 4 as a template for PCR using the conditions described above used. Genomic DNA from 11955 was used as a control. The PCR product (about 0.8 kb band was obtained from all three templates) was purified by agarose gel electrophoresis and eluted from the agarose gel using the QIAquick gel extraction kit. Samples were cut with NotI and electrophoresed on a 0.7% agarose gel to visualize the product. The 11955 PCR product does not show evidence of NotI cleavage, as expected, whereas the TM89 PCR product gives two bands around 0.4 kb, of the wild type gene with the mutated allele. Refers to substitution. NotI cleavage of the PCR product of TM15, the first integrant, gives two bands, mainly for the two bands seen in TM89 and for the minor uncleaved band (0.8 kb). This can be explained by the results obtained with Southern lots of TM15 genomic DNA.

  11955 genomic DNA, TM15 and TN89 were cut with NotI, PstI and NotI, and HindIII and NotI, and subjected to agarose gel electrophoresis. DNA was transferred to a positively charged nylon membrane (Roche) and hybridized with a DIG-labeled probe. The DIG-labeled probe was prepared by PCR of the 11955 LDH gene using primers 1 and 4 together with DIG-labeled dNTP according to the product instructions (Roche Molecular Biochemicals DIG Amplification Manual). Hybridized bands were visualized using a commercially available detection kit (Roche). Southern blots show evidence of a highly amplified band of about 7.5 kb at Not15 cleavage of TM15, similarly amplified bands of about 7 and 0.4 kb at TM15 cleavage of HindIII / NotI and PstI / NotI. Indication of incorporation of multiple tandem copies of pTM014 incorporated at the LDH position in one integrant. For all three restriction enzyme cuts, TM89 showed evidence of a different restriction enzyme pattern showing an additional hybridizing band compared to 11955, consistent with gene replacement. TM89 has been deposited with deposit number 41275 as described below.

Example 3
Reproducible growth of ethanol production and product production by wild type thermophile was achieved for the wild type thermophile in fed batch and continuous culture. Tables 1, 2 and 3 show the conditions used in the fermentation process.

Example 4
Lactic acid production was minimized through knockout of L-lactate dehydrogenase (LDH) by inactivation of the ldh gene to achieve ethanol yield and productivity, an increased target of ethanol production by thermophiles . Two approaches have been taken to inactivate the ldh gene: single crossover recombination of the marker DNA into the ldh region of the chromosome to prevent transcription, or create a mutant within that gene region. For this purpose, double recombination into the homologous region of the ldh gene of the DNA is to make the gene nonfunctional.
The single crossover approach quickly generated LDH negative mutants that showed increased ethanol production when compared to the wild type strain (NCIMB 11955).

Improvement of ethanol production by LDH mutants LDH negative mutants were grown in fed-batch culture in minimal medium made to measure the increase in ethanol production resulting from knockout of LDH activity. Changes in the metabolic profile of LDH mutants are shown in Table 5 and compared to the optimal ethanol production of the wild type strain. Metabolic production profiles of two LDH mutants are shown in FIGS. Table 6 shows the increase in ethanol production caused by the knockout of LDH activity.

  The LDH mutation produced significantly higher ethanol yields compared to the wild type, indicating that the metabolism of these thermophiles was successfully rerouted. Further optimization of the culture conditions and medium contents for the LDH mutant results in high ethanol yields.

Ethanol production by LDH mutants in continuous culture The most stable single crossed lactic acid mutant grows in continuous culture and evaluates its long-term stability as an ethanol producing bacterium. The strain was cultured in a minimal medium with glucose as a carbonate source. A steady culture was observed for about 290 hours before reverting to lactic acid production.

Example 5
Double cross mutant TM89 (Example 2) was assayed for ethanol production as described in Example 4. The results are shown in Table 8 and indicate that the mutant achieved significant levels of ethanol production (greater than 200 mM). Mutants are evaluated for ethanol production using glucose or xylose as a carbon source. The results are shown in Table 7.

  Glucose has been shown as the best carbon source, but this result clearly shows that the microorganism can ferment xylose without further modification.

The stability of lactate dehydrogenase negative mutants was tested under different conditions in continuous culture over a 1500 hour period. The results are shown in FIG. 6 and the lactate dehydrogenase negative phenotype remains stable despite considerable challenge to the culture and does not rely on antibiotic selection to remain in the phenotype It shows that. During this continuous run, dilution ratio will vary with 0.1hr ^-1 ~0.5hr ^-1, and changing the culture between aerobic (air) and anoxic (N ₂₎ Conditions However, there was no change in lactic acid concentration. More importantly, the pH of the culture was changed and lowered to pH 4.4, a pH that was outside the normal pH range of wild-type organism growth, but the culture changed in phenotype. Immediately returned to normal without showing. That is, the lactic acid concentration did not change.

  The microorganism defined herein as TM89 and the plasmid pUB190ldh were deposited under NCIMB accession numbers 41275 and 41276, respectively. The depositary institutions are NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, United Kingdom.

  The present invention will be described with reference to the accompanying drawings.

FIG. 1 is a graph showing ethanol production of the microorganism of the present invention (ldh35) using different substrates. FIG. 2 is a graph showing ethanol production of the microorganism of the present invention (ldh58) using different substrates. FIG. 3 is a graph showing ethanol production by the LDH knockout mutant, 11955. 4 and 5 are schematic diagrams of the pUB plasmid used in the present invention; FIG. 4 is an ldh mutation according to the present invention. 4 and 5 are schematic diagrams of the pUB plasmid used in the present invention; FIG. 4 is an ldh mutation according to the present invention. FIG. 6 is a graph showing the stability of LDH knockout mutants under different culture conditions.

Claims (21)

  A thermophilic microorganism modified to enable high ethanol production, wherein the modification is inactivation of a lactate dehydrogenase gene of a wild type thermophilic microorganism.   The microorganism according to claim 1, wherein the microorganism does not contain a restriction system.   The microorganism according to claim 1 or 2, wherein the microorganism is a Geobacillus species.   The microorganism according to any one of claims 1 to 3, wherein the microorganism is Geobacillus thermoglucosidasius.   The microorganism according to any one of claims 1 to 4, wherein the microorganism is a spore-forming bacterium.   6. The microorganism according to any one of claims 1 to 5, wherein the microorganism is stable in a culture medium containing up to 30% (w / v) ethanol.   The microorganism according to any one of claims 1 to 6, wherein the microorganism can metabolize cellobiose and / or starch, or an oligomer thereof.   The microorganism according to any one of claims 1 to 7, wherein the microorganism is transformed with high frequency.   The microorganism according to any one of claims 1 to 8, wherein the microorganism grows at a temperature of 40C to 85C, preferably 50C to 70C.   The microorganism according to any one of claims 1 to 9, wherein the microorganism comprises a non-natural pdc gene.   The microorganism according to any one of claims 1 to 10, wherein the microorganism comprises a non-natural adh gene.   The microorganism according to any one of claims 1 to 11, wherein the natural lactate dehydrogenase gene or a part thereof has been deleted.   The microorganism according to any one of claims 1 to 12, wherein the microorganism does not contain an integration element in a lactate dehydrogenase gene.   Microorganism deposited under NCIMB deposit number 41275. Production of ethanol comprising culturing the microorganism according to any one of claims 1 to 14 in a suitable situation in which C ₃ , C ₅ or C ₆ sugars or oligomers thereof are present. Method.   The method according to claim 15, wherein the method is performed at a temperature of 40 ° C. to 70 ° C.   The method of claim 16, wherein the temperature is from 52 ° C. to 65 ° C.   18. A method according to any one of claims 15 to 17, wherein the microorganism is maintained in a culture at a pH of 4 to 7.5.   A plasmid as defined herein as pUB190-ldh.   A thermophilic microorganism comprising the plasmid according to claim 19.   Animal feed containing the microorganism according to any one of claims 1 to 14.

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